Genotoxic agents exert their deleterious effects mainly by damaging cellular DNA. In response, cells have evolved several ways to overcome their harmful effects. One of the representative mechanisms is the repair of damaged DNA. Cells attempt to repair DNA damage before the onset of DNA replication or cell division. However, in the undesirable situation, replication of damaged DNA still occurs. Unrepaired DNA lesions often block the progression of DNA synthesis and are the major source of mutations. In this project, mechanisms for cellular responses to unrepaired DNA lesions will be studied using endogenously produced DNA adducts such as 1,N6-ethenodeoxyadenosine and one of the acrolein-derived deoxyguanosine adducts. These adducts are suspected to contribute to aging and cancer. Since they are continuously produced in cellular DNA, it is not unlikely that the cellular replication machinery encounters unrepaired endogenous lesions. If cells did not have any error-free damage tolerance mechanism, the survival and integrity of cellular DNA would depend solely on the efficiency and fidelity of translesion DNA synthesis, and a small number of blocking lesions would be lethal. However, many studies have shown that cells tolerate many unrepaired lesions. On the other hand, if cells had only error-free damage tolerance mechanism, cells would not be mutable by DNA adducts. However, cells are mutable by DNA adducts. Our central hypothesis is that cells respond to unrepaired DNA adducts in an error-free and an error-prone manner. Our preliminary studies have indicated that this is true in E. coli. This organism overcomes synthesis block by error-prone translesion synthesis and error-free daughter strand gap repair. We have demonstrated the existence of these two pathways, at the DNA sequence, using our recently developed approach. This approach utilizes a site-specifically placed single DNA adduct and strand-specific marker sequences to identify the origin of progeny which are derived from various cellular pathways. This new approach will be used to explore error-free and error-prone damage tolerance mechanisms in eukaryotes. The mechanisms will be investigated using plasmid and chromosomal substrates in human cells and yeast. The factors influencing damage tolerance mechanisms and their induction by DNA damage will also be investigated.